A preliminary screening demonstrated that the ethanolic extract of Eurycoma longifolia could decrease the basal level but increase the human chorionic gonadotropin (hCG)-induced production of testosterone by rat Leydig cells. Thirteen known compounds including five canthin-6-one alkaloids, three anthraquinones and five anthraquinone glucosides were isolated from the roots of E.longifolia. The structures of these compounds were established by spectroscopic analysis and comparison with literature data.